Sustained renewal of light-sensitive membranes, which comprise the rod outer segments (ROS) of retinal photoreceptors, is essential for their viability and for preservation of vision. Blinding eye diseases afflicting human populations are often caused by mutations that inactivate crucial components of the molecular machinery that regulates polarized membrane trafficking and ROS renewal in photoreceptor cells. In patients with retinitis pigmentosa, photoreceptor health is particularly compromised by the mutations in the C-terminal domain of rhodopsin. Our recent studies have revealed the existence of a sorting signal within this domain. This leads us to propose that the recognition of this signal initiates a cascade of molecular interactions that govern the targeted delivery of rhodopsin-carrying membranes to the ROS and associated morphogenic events. Using biochemical and cell biological approaches we have identified new candidates that regulate sequential steps in rhodopsin trafficking and ROS morphogenesis. Among them are the small GTPases of ARF, rab and rho families. We plan to study the role of these regulatory factors in rhodopsin trafficking and maintenance of photoreceptor polarity. We will delineate the interactions of rhodopsin with ARF GTPases and other regulatory proteins involved in polarized trafficking and photoreceptor membrane renewal. We will define the role in rhodopsin trafficking of rab11 and rad, small GTPases implicated by our preliminary results. To accomplish these goals, we will perturb membrane trafficking in healthy photoreceptors to model conditions found in patients with retinal degenerative diseases. We will modify, inactivate, or remove essential components of the sorting machinery, or introduce mutant proteins, and monitor their effects on membrane trafficking in vivo and in our established cell-free system in vitro. These studies will be complemented by morphological analysis of altered cells by confocal and electron microscopy. Our long-term goals are to better understand the molecular mechanisms by which mutations in rhodopsin and other photoreceptor genes trigger retinal degenerations. Ultimately, this knowledge will aid in the development of new strategies for treatment of retinal genetic disorders.